Project Summary In this proposal, we will develop new methods to overcome one of the main challenges in super-resolution microscopy and enable quantification of protein copy number at the nanoscale level. Super-resolution microscopy is an enabling tool that reveals the subcellular organization of molecular complexes with unprecedented spatial resolution. The nanoscopic organization of these complexes into functional units is highly important for regulating their subcellular activity in a spatially and temporally controlled manner. However, it has been very challenging to quantify the protein copy number composition of multi-protein complexes. Protein copy number is highly important for regulating or mis-regulating protein function. Proteins may be functional below a certain oligomeric assembly and gain toxic function when their oligomeric composition crosses a critical threshold leading to diseases. Therefore, the ability to properly quantify the sub- cellular copy number distribution of proteins within molecular complexes is important for gaining mechanistic insight into healthy and diseased function of these proteins. The main challenge, in doing so, is to overcome the artefacts arising from the unknown labeling stoichiometry and complex fluorophore photophysics. We have made important leaps towards overcoming this challenge by developing calibration nanotemplates for super-resolution microscopy. However, the lack of standard, easy-to-use methods and reagents that account for variability in experimental conditions has made it difficult for non-experts to adapt these developments. Therefore, there is an immediate need for highly standardized methodologies that allow protein copy number quantification independent of experimental conditions. The goal of this proposal is to address this big challenge and establish a versatile, easy-to-use and universal calibration method that can be adapted by the scientific community to quantify the copy number distribution of any protein of interest. Our aims are: (i) to develop calibration nanotemplates for both small and large protein complexes using DNA origami as well as novel nanotemplates that use designer protein nanocages, (ii) to acquire calibration data for diverse, super-resolution compatible labeling strategies and identify calibration functions, (iii) to develop the innovative concept of standardization based on novel use of benchmarking standards and methods that can transform the calibration functions among different experimental conditions and (iv) to develop an all-integrated, user-friendly, open- source, modular software that incorporates all the steps from single molecule localization to protein copy number determination. This proposal has the potential to advance super-resolution microscopy from a mainly descriptive tool into the era of quantitative and mechanistic cell biology. As one specific example, the methods developed here will make it possible to reveal the sub-cellular distribution and evolution of protein aggregation in a highly quantitative manner in several disease states at much earlier time points than has been possible thus far, potentially enabling new diagnostic and drug screening methods in the future. We expect the method to be widely applicable to a large number of biomedical questions and have a broad impact.